Pluripotent stem cells have tremendous potential to develop into different cell types. However, molecular mechanisms that endow stem cells with the capacity to maintain pluripotency or to differentiate into other cell types are not well understood. We found that inhibition of protein kinase C (PKC) isoforms by a single, selective PKC inhibitor maintains the undifferentiated phenotype of mouse embryonic stem (ES) cells without affecting their multidifferentiation potency. Furthermore, inhibition of PKC isoforms permits derivation of germline-competent ES cells from mouse blastocysts and also facilitates reprogramming of mouse embryonic fibroblasts towards induced pluripotent stem cells. The objective of this exploratory and developmental research proposal is to further validate the efficacy of the approach, in which PKC signaling pathway will be targeted to derive, propagate and maintain new ES cells from rat, to maintain pluripotency of human ES cells, and to understand associated molecular mechanisms. We will institute two specific aims in this proposal. In the first Specific Aim we will test the hypothesis that PKC signaling is an important pathway to dictate maintenance of pluripotency vs. differentiation of mammalian ES cells and, therefore, inhibition of PKC isoform function will help to derive, propagate and maintain pluripotent stem cells without affecting their complete developmental potential. We will test in vivo developmental potential of rat ES cells, derived and propagated with PKC inhibitors, by injecting them into the blastocysts to generate chimera and by assessing those chimeras for germline transmission. In addition, we will determine whether PKC inhibition maintains undifferentiated phenotypes of human ES cells and will assess pluripotency by developing teratomas in immune-compromised mice. In the second Specific Aim we will determine molecular mechanisms that are associated with PKC inhibition-mediated maintenance of stem cell pluripotency. We will test whether function of specific PKC isoform/s is important to induce mouse ES cell differentiation. In addition, we will also determine whether inhibition of PKC isoforms in ES cells maintains pluripotent stem cell-specific chromatin structure. We predict successful completion of this proposal will open up new areas of research to target PKC signaling pathway for stem cell biology and regenerative medicine purposes.